The baculovirus expression vector system (BEVS) is one of the most powerful and versatile eukaryotic expression systems. The BEVS is being used to produce antigens for vaccine development, to manufacture human therapeutics, to develop faster acting biological insecticides, and as a protein expression system for a multitude of research projects. However, a limitation of any lytic viral expression system, including BEVS, is that lysis of the infected cells interrupts protein expression and requires repetitive infections which results in decreased productivity levels and high production costs. ParaTechs and the Webb laboratory at the University of Kentucky have identified a gene family from an insect virus that delays death and lysis of baculovirus infected cells and significantly enhances recombinant protein production. This enhanced protein production presents a significant commercial opportunity that was the focus of ParaTechs' 'proof-of- concept' Phase I proposal. During Phase I, ParaTechs demonstrated utility and estimated the commercial value of its enhanced BEVS technology by quantifying the level of enhanced protein production and developing alternative methods of delivering the protein of interest to virus-infected cells. In this proposal we seek support to optimize enhancement of the technology in insect cell lines (objective 1), engineered baculovirus transfer viruses (objective 2) and in selectively modified baculovirus DNA (objective 3). We will quantify the effect of these modifications to ParaTechs' enhanced BEVS technology by measuring the expression of reporter, membrane-bound and secreted proteins. We have proven that ParaTechs' enhanced BEVS is more efficacious in the synthesis of some secreted proteins as the integrity of the cells infected with the enhanced BEVS is prolonged. A critical element for many modern medical treatments and research applications entails the ability to produce biologically active proteins with a high degree of safety. Baculovirus expression vectors have proven to have multiple advantages for protein production in a variety of applications such as production of proteins for vaccine development and human therapeutics for a number of diseases. The enhanced BEVS technology to be developed with this award has potential to significantly improve efficacy of protein production in all such medical applications. [unreadable] [unreadable] [unreadable]